alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme - The resulting Neu5Aece2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha2,3-sialylated GalNAc residu es have been reported. In the present experiments, recombinant soluble alph a2,3-sialyltransferase ST3Gal II efficiently sialylated the X-2 pentasaccha ride GalNAc beta1-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc, globo-N-tetraos e GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and the disaccharide GalNAc bet a1-3Gal in vitro. The purified products were identified as Neu5Ac alpha2-3G alNAc beta1-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc, Neu5Ac alpha2-3GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and Neu5Ac alpha2-3GalNAc beta1-3Gal, r espectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR -spectroscopy. In particular, the presence of the Neu5Ac alpha2-3GalNAc lin kage was firmly established in all three products by a long range correlati on between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlati on spectra. Collectively, the data describe the first successful sialyltran sfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3 Gal II has been shown to transfer to the Gal beta1-3GalNAc determinant. Con sequently, the present data show that the enzyme is multifunctional, and co uld be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X2 pentasaccharide. The Neu5Ac alpha2-3GalNAc linkage of s ialyl X2 was cleaved by sialidases from Arthrobacter ureafaciens and Clostr idium perfringens, but resisted the action of sialidases from Newcastle dis ease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha2-3GalNAc and Neu5Ac al pha2-6GalNAc linkages, as has been assumed previously.